In general, molecular analyses of mammalian cell functions, such as gene transcription and its regulation, have been carried out with cells that have suffered neoplastic transformation or have been otherwise converted to continuously dividing, established cell lines. Although much valuable information has been extracted from these experimentally tractable models, it is clear that normal and immortalized cells differ in important ways. Defining and understanding these differences at the molecular level is a goal in understanding normal cell function, and of cancer and other proliferative diseases.
The conversion of a normal cell into a neoplastic cell occurs in multiple steps (Vogelstein and Kinzler (1993) Trends Genet., 9: 138-141). One approach to studying this process employs transgenie mice carrying dominant negative oncogenes or inactivated tumor suppressor genes (Hanahan (1988) Annu. Rev. Genet., 22: 479-519). For example, a small proportion of dermal fibroblasts in mice bearing transgenie bovine papillomavirus type I (BPV-1) genomes proceeds through two histological grades of hyperplasia, termed mild and aggressive fibromatosis, and finally emerges as dermai fibrosarcomas. Cells cultured from each of these stages appear to retain characteristics of the lesions from which they were derived (Sippola-Thiele et al. (1989) Mol. Cell. Biol. 9: 925-934). All three pathological stages contain BPV-1 DNA and RNA transcripts, and the aggressive fibromatosis and fibrosarcoma cells form tumors after inoculation into mice. Importantly, the aggressive fibromatosis and fibrosarcoma cultures contain similar levels of the BPV-1 E5 and E6 oncogene products (Sippola-Thiele et al., Supra). Thus, the BPV-1 transgene is not a sufficient determinant of the dermal fibrosarcoma phenotype, suggesting that changes in cellular components must contribute to the conversion of the advanced hyperplasia into fibrosarcoma.
Fibrosarcomas contain one or both of two karyotypic defects, neither of which is seen in the fibromatosis stages: translocations involving chromosome 14 (60%), or duplications of chromosome 8 (70%); about 30% of the tumors carry both lesions (Lindgren et al., (1989) Proc. Natl Acad. Sci. USA 86:5025-5029). It is apparent that sites of consistent chromosome rearrangements can localize genes that may be critically involved in malignant transformation, and that the rearrangements themselves can subvert the normal functioning of these genes (Bishop (1991) Cell, 64:235-248; Solomon et al., (1991) Science, 254:1153-1160; Marshall (1991) Cell, 64:313-326). In this regard, it seemed potentially interesting that a proto-oncogene, JunB, was found to reside within a region of chromosome 8 (Mattei et al. (1990) Oncogene, 5: 151-156) that is most commonly duplicated in fibrosarcomas (Lindgren et al., Supra). A survey of several members of the AP-1 factor family (Vogt et al. (1990) Adv. Cancer Res., 55: 1-35; Hunter et al. (1991) Cell, 64:249-270; Hunter and Karin (1992) Cell, 70:375-387; Kerppola and Curran (1991) Science, 254:1210-1214) during the progression to fibrosarcoma revealed that JuneB and c-Jun were elevated in the aggressive fibromatosis as well as fibrosarcoma cultures, whereas JunD and c-Fos remained constant (Bossy-Wetzel et al. (1992) Genes and Dev., 6:2340-2351). However, overexpression of JuneB and/or c-Jun was not sufficient to induce the complete tumor cell phenotype; mild fibromatosis cells overexpressing either or both of these genes displayed anchorage-independent growth in soft agar, but failed to produce tumors upon inoculation into histocompatible mice. Thus, it appears that additional events during tumor progression distinguish aggressive fibromatoses from fibrosarcomas.
Extensive interactions have been described between AP-1 factors and various members of the "intracellular receptor" super family, including the estrogen receptor (ER) (Gaub et al. (1990) Cell, 63:1267-1276; Doucas et al. (1991), EMBO J., 10:2237-2245), the retinoic acid (RAR) and vitamin D (VDR) receptors (Schuele et al. (1990a) Cell 61:497-504; Schuele et aI. (1990b) Cell, 62:1217-1226), and especially the glucocorticoid receptor (GR) (Diamond et al. (1990) Science, 249: 1266-1272; Jonat et al. (1990) Cell 62:1189-1204; Schuele et al, (1990b)Supra; Yang et aL (1990) Cell, 62: 1205-1215). The intracellular receptors mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D (for reviews see Evans (1988) Science, 240:889-895; Ham and Parker (1989) Curr. Opin. Cell Biol., 1:503-511; Bumstein et al. (1989), Ann. Rev. Physiol., 51:683-699; Truss and Beato (1993) Endocr. Rev., 14:459-479). Although distinct in detail, these receptors share general characteristics of structure and mode of action. Thus, the GR binds its cognate hormone in the cytoplasm, migrates to the nucleus and regulates transcription upon association with specific glucocorticoid response element (GRE) DNA sequences near target genes. Two broad classes of GREs have been described: GR binding at "simple GREs", which contain imperfect palindromes of hexamer half-sites separated by three base pairs (Beato (1989) Cell, 56:335-344), is sufficient for enhancement of transcription from nearby promoters, although repression has not been observed from such sites. In contrast, GR acts only in collaboration with other factors that bind at "composite GRE" sites that lack a common consensus sequence; at these sites, GR can either enhance or repress transcription (Yamamoto et al., (1992) In McKnight and Yamamoto (eds.) Transcriptional regulation Cold Spring Harbor Lab. Press, New York, 1169-1192).
Steroid hormones are essential regulators of normal cell growth, differentiation and homeostasis (for review see Walsh and Avashia (1992) Cleveland Clinic J. Med. 59:505-515). For example, estrogens and androgens can function as powerful mitogens while also playing critical roles in differentiation (Jordan and Morrow (1993) Stem Cells 11:252-262; Wilding (1992) Cancer Surv. 14:113-130), whereas glucocorticoids tend to promote differentiation and inhibit proliferation. Steroids are also commonly used therapeutics. Thus, topical glucocorticoids reduce tissue destruction in certain skin disorders by down-regulating type IV collagenase (Oikarinen et aI., (1993) J. Invest. Dermatol. 101:205-210). Intracellular receptors and their ligands have been also implicated in various malignancies. For example, the RAR.beta. gene can be rearranged as a result of hepatitis B virus integration in certain human hepatocellular carcinomas (Dejean et al. (1986) Nature, 322:70-72), and RAR.alpha. is split into two chimeric proteins by a t(15:17) translocation in acute promyelocyfic leukemia (De The et al. (1990) Nature, 347:558-561; Borrow et al. (1990) Science, 249:1577-1580).
Estrogens promote the course of several cancers (Jordan and Murphy (1990) Endocr. Rev., 11:578-610). Glucocorticoids inhibit the growth of carcinogen-induced tumors in mouse lung (Droms et al. (1993), Int. J. Cancer, 53: 1017-1022), mouse skin (Strawhecker and Pelling (1992) Carcinogenesis, 13:2075-2080) and rat colon (Denis et al. (1992), J. Steroid Blochem. and Mol. Biol., 41:739-745). In contrast, glucocorticoids markedly enhance the transformation of cultured human epithelial cells by Kirsten murine sarcoma virus (KMSV) (Durst et al. (1989) Virology, 173:767-771), and strongly activate mouse mammary tumor virus gene transcription and virus production (Truss and Beato (1993) Supra).
The present invention demonstrates for the first time that the activity of intracellular hormones differs between normal and immortalized cells, explaining various aspects of the functional interactions of intracellular receptors with AP1, in addition to the effects of steroids and retinoids on cell growth and differentiation.